The Impact of Chemical Modifications on the Interferon-Inducing and Antiproliferative Activity of Short Double-Stranded Immunostimulating RNA.

A short 19 bp dsRNA with 3'-trinucleotide overhangs acting as immunostimulating RNA (isRNA) demonstrated strong antiproliferative action against cancer cells, immunostimulatory activity through activation of cytokines and Type-I IFN secretion, as well as anti-tumor and anti-metastatic effects in vivo. The aim of this study was to determine the tolerance of chemical modifications (2'-F, 2'-OMe, PS, cholesterol, and amino acids) located at different positions within this isRNA to its ability to activate the innate immune system. The obtained duplexes were tested in vivo for their ability to activate the synthesis of interferon-α in mice, and in tumor cell cultures for their ability to inhibit their proliferation. The obtained data show that chemical modifications in the composition of isRNA have different effects on its individual functions, including interferon-inducing and antiproliferative effects. The effect of modifications depends not only on the type of modification but also on its location and the surrounding context of the modifications. This study made it possible to identify leader patterns of modifications that enhance the properties of isRNA: F2/F2 and F2_S/F2 for interferon-inducing activity, as well as F2_S5/F2_S5, F2-NH2/F2-NH2, and Ch-F2/Ch-F2 for antiproliferative action. These modifications can improve the pharmacokinetic and pharmacodynamic properties, as well as increase the specificity of isRNA action to obtain the desired effect.

Influence of the Composition of Cationic Liposomes on the Performance of Cargo Immunostimulatory RNA.

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, ß, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or ß, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.

Activation of Innate Immunity by Therapeutic Nucleic Acids.

Nucleic acid-based therapeutics have gained increased attention during recent decades because of their wide range of application prospects. Immunostimulatory nucleic acids represent a promising class of potential drugs for the treatment of tumoral and viral diseases due to their low toxicity and stimulation of the body's own innate immunity by acting on the natural mechanisms of its activation. The repertoire of nucleic acids that directly interact with the components of the immune system is expanding with the improvement of both analytical methods and methods for the synthesis of nucleic acids and their derivatives. Despite the obvious progress in this area, the problem of delivering therapeutic acids to target cells as well as the unresolved issue of achieving a specific therapeutic effect based on activating the mechanism of interferon and anti-inflammatory cytokine synthesis. Minimizing the undesirable effects of excessive secretion of inflammatory cytokines remains an unsolved task. This review examines recent data on the types of immunostimulatory nucleic acids, the receptors interacting with them, and the mechanisms of immunity activation under the action of these molecules. Finally, data on immunostimulatory nucleic acids in ongoing and completed clinical trials will be summarized.

Evolution of Tandemly Arranged Repetitive DNAs in Three Species of Cyprinoidei with Different Ploidy Levels.

Polyploid species represent a challenge for both cytogenetic and genomic studies due to their high chromosome numbers and the morphological similarity between their paralogous chromosomes. This paper describes the use of low-coverage high-throughput sequencing to identify the 14 most abundant tandemly arranged repetitive elements in the paleotetraploid genome of the crucian carp (Carassius carassius, 2n = 100). These repetitive elements were then used for molecular cytogenetic studies of a closely related functionally triploid form of the Prussian carp (Carassius gibelio, 3n = 150 + Bs) and a relatively distant diploid species, the tench (Tinca tinca, 2n = 48). According to their distribution on the chromosomes of the 3 aforementioned species, the repetitive elements here identified can be divided into 5 groups: (1) those specific to a single genomic locus in both Carassius species, despite the recent carp-specific genome duplication; (2) those located in a single genomic locus of T. tinca, but amplified in one or both Carassius species; (3) those massively amplified in the B chromosomes of C. gibelio; (4) those located in a single locus in C. gibelio, but amplified in many blocks in C. carassius; and (5) those located in multiple pericentromeric loci in both Carassius species. Our data indicate that some of the repetitive elements are highly conserved in cyprinoid species and may serve as good cytogenetic and genomic markers for discriminating paralogous chromosomes, while others are evolutionarily recent, and their amplification may be related to the last whole-genome duplication event.